The present invention relates to novel compounds and methods of preparation thereof and, particularly, to E-ring expanded camptothecin derivatives or analogs and to methods of preparation of such camptothecin analogs.
Camptothecins are DNA topoisomerase I inhibitors now being used as anticancer drugs. Topotecan (tpt) and CPT-11 are the first two members in the camptothecin family to gain Food and Drug Administration full approval status (topotecan in 1996 as second-line therapy for advanced epithelial ovarian cancer, topotecan again in 1998 for the treatment of small cell lung cancer, CPT-11 in 1998 as first-line therapy for colon cancer). Several other analogs of the camptothecin family such as GI-147211C, DX8951f, 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin are in various stages of pre-clinical and clinical evaluation. Each of the campothecins in clinical use undergoes relatively rapid hydrolysis in the bloodsteam resulting in a marked loss of anticancer activity. It is the key xcex1-hydroxylactone pharmacophore within clinically relevant camptothecins that hydrolyzes at physiological pH to yield a biologically-inactive and potentially toxic hydroxy carboxylate form. Fassberg, J. and Stella, V. J., xe2x80x9cA Kinetic and Mechanistic Study of the Hydrolysis of Camptothecin and Some Analoguesxe2x80x9d, J. Pharm. Sci. 81: 676-684 (1992); Hertzberg, R. P., Caranfa, M. J., and Hecht, S. M., xe2x80x9cOn the Mechanism of Topoisomerase I Inhibition by Camptothecin: Evidence for Binding to an Enzyme-DNA Complexxe2x80x9d, Biochemistry 28: 4629-4638 (1989); Hsiang, Y-H., and Liu, L. F., xe2x80x9cIdentification of Mammalian DNA Topoisomerase I as an Intracellular Target of the Anticancer Drug Camptothecinxe2x80x9d, Cancer Res. 48: 1722-1726 (1988); and Jaxel, C., Kohn, K. W., Wani, M. C., Wall, M. E., and Pommier, Y., xe2x80x9cStructure-Activity Study of Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for a Specific Receptor Site and a Relation to Antitumor Activityxe2x80x9d, Cancer Res. 49: 5077-5082 (1989). References set forth herein, including those set forth above, may facilitate understanding of the present invention. Inclusion of a reference herein is not intended to an does no constitute an admission that the reference is prior art with respect to the present invention.
The structures of camptothecin and some of its important analogs are shown below: 
Recent research efforts have shown that agents such as 9-aminocamptothecin and camptothecin (cpt) display very poor stabilities in human blood due to high affinity binding interactions between their carboxylate forms and human serum albumin (HSA). Burke, T. G, Mi, Z., Jiang, Y., and Munshi, C. B. xe2x80x9cThe Important Role of Albumin in Determining the Relative Human Blood Stabilities of the Camptothecin Anticancer Drugsxe2x80x9d, Journal of Pharmaceutical Sciences, 84: 518-519 (1995); Burke, T. G. and Mi, Z. xe2x80x9cThe Structural Basis of Camptothecin Interactions with Human Serum Albumin: Impact on Drug Stabilityxe2x80x9d, Journal of Medicinal Chemistry, 37: 40-46 (1994); Mi, Z. and Burke, T. G., xe2x80x9cDifferential interactions of Camptothecin Lactone and Carboxylate Forms with Human Blood Componentsxe2x80x9d, Biochemistry, 33: 10325-10336 (1994); and Mi, Z., Malak, H., and Burke, T. G. xe2x80x9cReduced Albumin Binding Promotes the Stability and Activity of Topotecan in Human Bloodxe2x80x9d, Biochemistry, 34: 13722-13728 (1995), the disclosures of which are incorporated herein by reference. Frequency-domain lifetime fluorometry experiments revealed that human serum albumin (HSA) preferentially binds camptothecin carboxylate with over a 100-fold higher affinity compared to camptothecin lactone. Mi, Z. and Burke, T. G. xe2x80x9cMarked Interspecies Variations Concerning the Interactions of Camptothecin with Serum Albumins: A Frequency-Domain Fluorescence Spectroscopic Studyxe2x80x9d, Biochemistry, 33: 12540-12545 (1994), the disclosure of which is incorporated herein by reference. This differential binding of carboxylate over lactone results in camptothecin and 9-AC opening more rapidly and completely in the presence of HSA than in the absence of the protein. In human plasma, pH 7.4 and 37xc2x0 C., camptothecin and 9-AC both open rapidly and essentially completely to almost negligible 0.2% lactone levels at equilibrium. While the presence of HSA promotes lactone ring opening for camptothecin and 9-AC, red blood cells and lipid bilayers in general preferentially bind the electroneutral lactone forms of camptothecins over their respective negatively-charged carboxylate lactone forms. Burke, T. G., Staubus, A. E., Mishra, A. K., and Malak, H., xe2x80x9cLiposomal Stabilization of Camptothecin""s Lactone Ringxe2x80x9d, J. Am. Chem. Soc. 114: 8318-8319 (1992); and Burke, T. G., Mishra, A. K., Wani, M., and Wall, M., xe2x80x9cLipid Bilayer Partitioning and Stability of Camptothecin Drugsxe2x80x9d, Biochemistry, 32: 5352-5364 (1993), the disclosures of which are incorporated herein by reference. Drug interactions with erythrocytes thereby promote active lactone levels in blood. 
Recently, Lavergne et al. have shown that expansion of the E-ring of camptothecin to produce a xe2x80x9chomocamptothecinxe2x80x9d enhances the solution stability of camptothecin while maintaining anticancer activity. Lavergne, O., Lesueur-Ginot, L., Rodas, F. P., Kasprzyk, P. G., Pommier, J., Demarquay, D., Prevost, G., Ulibarri, G., Rolland, A., Schiano-Liberatore, A.-M., Harnett, J., Pons, D., Camara, J., Bigg, D., xe2x80x9cHomocamptothecins: Synthesis and Antitumor Activity of Novel E-Ring Modified Camptothecin Analogsxe2x80x9d, J. Med. Chem., 41, 5410-5419 (1998); and Lavergne, O., Lesueur-Ginot, L., Rodas, F. P., and Bigg, D., xe2x80x9cAn E-Ring Modified Camptothecin With Potent Antiproliferative and Topoisomerase I inhibitory Activities. Bioorg. Med. Chem. Lett. 7, 2235-2238 (1997). The modification to the E-ring in the studies of Lavergne et al. involved insertion of a methylene spacer between the 20-OH functionality and the carboxyl group of the naturally occurring six-membered xcex1-hydroxylactone of camptothecin. Incorporation of the new 7-membered xcex2-hydroxylactone ring into camptothecin was found to improve the solution and plasma stability of the agent.
The structure of the homocamptothecin of Lavergne et al. and the numbering system used to describe such compounds are shown below: 
Although substantial strides have been made in the in development of the camptothecin family of drugs, it remains very desirable to develop improved compounds in this family of drugs and to develop improved synthetic routes for producing such drugs.
The present invention provides generally for a compound having the following formula (1): 
in racemic form, enantiomerically enriched form or enantiomerically pure form;
wherein R1 and R2 are independently the same or different and are hydrogen, xe2x80x94C(O)Rf wherein Rf is an alkyl group, an alkoxy-group, an amino group or a hydroxy group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryloxy group, an acyloxy group, xe2x80x94OC(O)ORd, wherein Rd is an alkyl group, xe2x80x94OC(O)NRaRb wherein Ra and Rb are independently the same or different, H, xe2x80x94C(O)Rf, an alkyl group or an aryl group, a halogen, a hydroxy group, a nitro group, a cyano group, an azido group, a formyl group, a hydrazino group, an amino group, xe2x80x94SRc, wherein Rc is hydrogen, xe2x80x94C(O)Rf, an alkyl group or an aryl group; or R1 and R2 together form a chain of three or four members selected from the group of CH, CH2, O, S, NH, or NR15, wherein R15 is an C1-C6 alkyl group;
R3 is H, a halogen atom, a nitro group, an amino group, a hydroxy group, or a cyano group; or R2 and R3 together form a chain of three or four members selected from the group of CH, CH2, O, S, NH, or NR15, wherein R15 is an C1-C6 alkyl group;
R4 is H, F, an amino group, a C1-3 alkyl group, a C2-3 alkenyl group, a C2-3 alkynyl group, a trialkylsilyl group or a C1-3 alkoxy group;
R5 is a C1-10 alkyl group, an alkenyl group, an alkynyl group, or a benzyl group;
R6 is xe2x80x94Si(R8R9R10) or xe2x80x94(R7)Si(R8R9R10), wherein R7 is an alkylene group, an alkenylene group, or an alkynylene group;
and R8, R9 and R10 are independently a C1-10 alkyl group, a C2-10 alkenyl group, a C2-10 alkynyl group, an aryl group or a xe2x80x94(CH2)NR11 group, wherein N is an integer within the range of 1 through 10 and R11 is a hydroxy group, an alkoxy group, an amino group, an alkylamino group, a dialkylamino group, a halogen atom, a cyano group, xe2x80x94SRc or a nitro group;
R13 is H, F or xe2x80x94CH3;
R16 is xe2x80x94C(O)Rf or H; and
pharmaceutically acceptable salts thereof.
R1 and R2 together may, for example, form a group of the formula xe2x80x94O(CH2)nOxe2x80x94 wherein n represents the integer 1 or 2. Likewise, R2 and R3 together may, for example, form a group of the formula xe2x80x94O(CH2)nOxe2x80x94 wherein n represents the integer 1 or 2.
R5 is preferably an ethyl group, an allyl group, a benzyl group or a propargyl group. Most preferably, R5 is an ethyl group. Preferably, R4 is H.
In one embodiment, R8 and R9 are methyl groups, R10 is a tert-butyl group or a methyl group, R1 is H and R3 is H. In this embodiment, R2 may, for example, be H, NH2 or OH.
R13 is preferably H. R16 is preferably H or an alkyl group. Most preferably, R16 is H or xe2x80x94C(O)Rf, wherein Rf is an alkyl group. Most preferably, R16 is H.
The present invention also provides a method of synthesizing a compound having the formula 
via a cascade radical 4+1 annulation wherein the precursor 
or the precursor 
is reacted with an arylisonitrile having the formula 
wherein X is a radical precursor. Preferably, X is Cl, Br or I. Most preferably, X is Br or I.
The present invention also provides a compound having the formula 
in racemic form, enantiomerically enriched form or enantiomerically pure form, wherein R12 is preferably H or xe2x80x94C(O)Rf, xe2x80x94C(O)ORd or xe2x80x94C(O)NRaRb; and
pharmaceutically acceptable salts thereof.
The present invention further provides compounds having the formulas 
in racemic form, enantiomerically enriched form or enantiomerically pure form;
Still further, the present invention provides a compound having the formula 
in racemic form, enantiomerically enriched form or enantiomerically pure form.
The present invention also provides a compound having the formula 
The present invention also provides a compound having the formula 
wherein R15 is a C1-C6 alkyl group.
The present invention further provides a compound having the formula 
in racemic form, enantiomerically enriched form or enantiomerically pure form, wherein R14 is SiMe3, I, or Br.
Still further, the present invention provides a method of synthesizing a compound having the following formula: 
wherein Y is chlorine, bromine or iodine;
comprising the steps of
(a) treating an enol ether of the structure: 
xe2x80x83under suitable oxidative cleavage conditions to form a compound having the structure: 
(b) treating the compound formed in step (a) with an organometallic reagent having the structure:
MC(R13)(R13)CO2R15
xe2x80x83wherein M is Li, Na, K, MgY, or ZnY under suitable conditions to form a compound having the structure: 
(c) treating the compound formed in step (b) under suitable conditions with acid to form a compound having the structure: 
(d) treating the compound formed in step (c) under suitable conditions of halogenative desilylation to form a compound having the structure: 
(e) treating the compound in step (d) with acid or iodottimethylsilane under suitable conditions for demethylation to provide a compound of the following structure: 
(f) treating the compound in step (e) with a lithium base or a sodium base in the presence of an inorganic lithium salt to deprotonate the nitrogen atom,
(g) reacting of the resulting deprotonated species of step
(f) with a compound of the following structure: 
xe2x80x83wherein Z is I, Br, Cl, a mesylate group or a tosylate group, and under suitable conditions to cause the formation of the compound of the following structure: 
As indicated above, all compounds of the present invention including the xcex2-hydroxylactone group can exist in racemic form, enantiomerically enriched form, or enantiomerically pure form. The formulas of such compounds as set forth herein cover and/or include each such form.
The term xe2x80x9cradical precursor(s)xe2x80x9d as used herein and as well known to those skilled in the art refers generally to those functional groups that cleave to generate radicals under standard conditions of chain or non-chain radical reactions. Common radical precursors are the halogens (except fluorine), carboxylic acids and derivatives thereof (such as thiohydroxamates), selenophenyl groups, diazonium salts, and the like. See, for example, Giese, B. Radicals in Organic Synthesis: Formation of Carbon-Carbon Bonds; Pergamon, Oxford (1986), the disclosure of which is incorporated herein by reference.
The terms xe2x80x9calkylxe2x80x9d, xe2x80x9carylxe2x80x9d and other groups refer generally to both unsubstituted and substituted groups unless specified to the contrary. Unless otherwise specified, alkyl groups are hydrocarbon groups and are preferably C1-C15 (that is, having 1 to 15 carbon atoms) alkyl groups, and more preferably C1-C10 alkyl groups, and can be branched or unbranched, acyclic or cyclic. The above definition of an alkyl group and other definitions apply also when the group is a substituent on another group (for example, an alkyl group as a substituent of an alkylamino group or a dialkylamino group). The term xe2x80x9carylxe2x80x9d refers to phenyl or naphthyl. As used herein, the terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refer to fluoro, chloro, bromo and iodo.
The term xe2x80x9calkoxyxe2x80x9d refers to xe2x80x94ORd, wherein Rd is an alkyl group. The term xe2x80x9caryloxyxe2x80x9d refers to xe2x80x94ORe, wherein Re is an aryl group. The term acyl refers to xe2x80x94C(O)Rf. The term xe2x80x9calkenylxe2x80x9d refers to a straight or branched chain hydrocarbon group with at least one double bond, preferably with 2-15 carbon atoms, and more preferably with 2-10 carbon atoms (for example, xe2x80x94CHxe2x95x90CHRg or xe2x80x94CH2CHxe2x95x90CHRg). The term xe2x80x9calkynylxe2x80x9d refers to a straight or branched chain hydrocarbon group with at least one triple bond, preferably with 2-15 carbon atoms, and more preferably with 2-10 carbon atoms (for example, xe2x80x94Cxe2x89xa1CRh or xe2x80x94CH2xe2x80x94Cxe2x89xa1CRh). The terms xe2x80x9calkylene,xe2x80x9d xe2x80x9calkenylenexe2x80x9d and xe2x80x9calkynylenexe2x80x9d refer to bivalent forms of alkyl, alkenyl and alkynyl groups, respectively.
The groups set forth above, can be substituted with a wide variety of substituents to synthesize homocamptothecin analogs retaining activity. For example, alkyl groups may preferably be substituted with a group or groups including, but not limited to, a benzyl group, a phenyl group, an alkoxy group, a hydroxy group, an amino group (including, for example, free amino groups, alkylamino, dialkylamino groups and arylamino groups), an alkenyl group, an alkynyl group and an acyloxy group. In the case of amino groups (xe2x80x94NRaRb), Ra and Rb are preferably independently hydrogen, an acyl group, an alkyl group, or an aryl group. Acyl groups may preferably be substituted with (that is, Rf is) an alkyl group, a haloalkyl group (for example, a perfluoroalkyl group), an alkoxy group, an amino group and a hydroxy group. Alkynyl groups and alkenyl groups may preferably be substituted with (that is, Rg and Rh are preferably) a group or groups including, but not limited to, an alkyl group, an alkoxyalkyl group, an amino alkyl group and a benzyl group.
The term xe2x80x9cacyloxyxe2x80x9d as used herein refers to the group xe2x80x94OC(O)Rd.
The term xe2x80x9calkoxycarbonyloxyxe2x80x9d as used herein refers to the group xe2x80x94OC(O)ORd.
The term xe2x80x9ccarbamoyloxyxe2x80x9d as used herein refers to the group xe2x80x94OC(O)NRaRb.
Amino and hydroxy groups may include protective groups as known in the art. Preferred protective groups for amino groups include tert-butyloxycarbonyl, formyl, acetyl, benzyl, p-methoxybenzyloxycarbonyl, trityl. Other suitable protecting groups as known to those skilled in the art are disclosed in Greene, T., Wuts, P. G. M., Protective Groups in Organic Synthesis, Wiley (1991), the disclosure of which is incorporated herein by reference.
In general, R1, R2, R3, R6, R7 and R8 are preferably not excessively bulky to maintain activity of the resultant camptothecin analog. Preferably, therefore, R1, R2, R3, R6, R7 and R8 independently have a molecular weight less than approximately 250. More preferably R1, R2, R3, R6, R7 and R8 independently have a molecular weight less than approximately 200.
Some of the camptothecin analogs of the present invention can be prepared for pharmaceutical use as salts with inorganic acids such as, but not limited to, hydrochloride, hydrobromide, sulfate, phosphate, and nitrate. The camptothecin analogs can also be prepared as salts with organic acids such as, but not limited to, acetate, tartrate, fumarate, succinate, citrate, methanesulfonate, p-toluenesulfonate, and stearate. Other acids can be used as intermediates in the preparation of the compounds of the present invention and their pharmaceutically acceptable salts.
For purification, administration or other purposes, the E-ring (the lactone ring) may be opened with alkali metal such as, but not limited to, sodium hydroxide or calcium hydroxide, to form opened E-ring analogs of compounds of formula (1) as set forth in the compounds of formula (2). The intermediates thus obtained are more soluble in water and may be purified to produce, after treatment with an acid, a purified form of the camptothecin analogs of the present invention.
The E-ring may also be modified to produce analogs of compounds of formula (1) with different solubility profiles in water or other solvents. Methods to achieve this goal include, but are not limited to, opening the E-ring with hydroxide or a water-soluble amino group or functionalizing the hydroxy group at position 20 of the E-ring with a water-soluble group such as a polyethylene glycol group or an acyl group. Such groups can be introduced either on the homocamptothecin derivative or at an earlier stage in the synthesis. The analogs thus prepared act as pro-drugs. In other words, these analogs regenerate the compounds of formula (1) (with the closed E-ring structure) when administered to a living organism. See, Greenwald, R. B. et al., J. Med. Chem., 39, 1938 (1996). Alkyl esters resulting from acylation at C20, for example, will result in more lipophilic pro-drugs that may not hydrolyze until the alkyl group is enzymatically cleaved.
The present invention also provides a method of treating a patient, which comprises administering a pharmaceutically effective amount of a compound of formulas (1) and/or (2) or a pharmaceutically acceptable salt thereof. The compound may, for example, be administered to a patient afflicted with cancer and/or leukemia. The compounds of the present invention may also act as antiviral (for example, anti-HIV) agents and antiparasitic agents. The compounds of formulas (1) and/or (2) may be administered by any conventional route of administration, including, but not limited to, intravenously, intramuscularly, orally, subcutaneously, intratumorally, intradermally, and parenterally. The pharmaceutically effective amount or dosage is preferably between 0.01 to 60 mg of one of the compounds of formulas (1) and (2) per kg of body weight. More preferably, the pharmaceutically effective amount or dosage is preferably between 0.1 to 40 mg of one of the compounds of formulas (1) and (2) per kg of body weight. In general, a pharmaceutically effective amount or dosage contains an amount of one of the compounds of formulas (1) and/or (2) effective to display antileukemic, antitumor (anticancer), antiviral and/or antiparisitic behavior. Pharmaceutical compositions containing as an active ingredient one of the compounds of formulas (1) and/or (2) or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable carrier or diluent are also within the scope of the present invention.
The present invention also provides a pharmaceutical composition comprising any of the compounds of formulas (1) and (2) and a pharmaceutically acceptable carrier. The composition may, for example, contain between 0.1 mg and 3 g, and preferably between approximately 0.1 mg and 500 mg of the compounds of formulas (1) and/or (2), and may be constituted into any form suitable for the mode of administration.
The structural modifications of the present invention were found to prevent high affinity binding between the carboxylate form of a camptothecin analog and HSA, while at the same time promoting lactone interactions with erythrocytes. An additional consideration in the design of plasma and blood-stable camptothecins concerns the structure of the E-ring. The A,B,E- or B,E-ring modified camptothecins of the present invention: 1) display enhanced stability in the presence of HSA through elimination or minimization of the highly preferential binding by HSA of carboxylate over lactone forms; 2) display high levels of lipophilicity which promote reversible associations of the lactone forms of the drugs with red blood cells, thereby slowing and restricting the extent of drug hydrolysis; and 3) display improved stability in aqueous solution.
We further discovered that the novel blood-stable silyl-substituted homocamptothecin (referred to herein as xcex2-hydroxylactone silatecans or homosilatecans (hST)) derivatives of the present invention can be prepared by significant modification of a total synthesis approaches set forth in U.S. patent application Ser. No. 09/212,178, entitled CAMPTOTHECIN ANALOGS AND METHODS OF PREPARATION THEREOF and filed Dec. 15, 1998 and U.S. patent application Ser. No. 09/007,872, entitled NOVEL INTERMEDIATES IN THE SYNTHESIS OF CAMPTOTHECIN AND RELATED COMPOUNDS AND SYNTHESIS THEREOF and filed Jan. 15, 1998 the disclosure of which are incorporated herein by reference. Novel intermediates were synthesized to carry out the cascade radical annulation of the present invention.
Several model compounds of the present invention, as described in the formula below, were studied extensively. 
The novel homocamptothecins of the present invention contain A,B- or B-ring modifications which decrease the preferential carboxylate over lactone binding by human albumin. These modifications in the A,B-rings also markedly enhance lipophilicity and promote lactone associations with lipid bilayers present in blood. The new compounds also contain an expanded xcex2-hydroxylactone E-ring which improved the overall stability of the agents without loss of potency. In cytotoxicity assays using MDA-MB-435 breast cancer cells, the E-ring expanded xcex2-hydroxylactone silatecans of the present invention display IC50 values in the range of 2 to 115 nM. The compounds of the present invention (several of which are described in the formula above), as a result of their novel structural substitutions, have superior human plasma and human blood stabilities than the agents described by Lavergne et al.
Synthesis of the novel A,B,E-ring modified and B,E-ring modified camptothecins of the present invention has led to the identification of the most blood-stable camptothecins displaying intrinsic potency yet to be identified. An additional benefit of these new agents is that they do not display any significant interspecies variations in blood stabilities such as those of 9-AC and camptothecin described in Mi, Z. and Burke, T. G. xe2x80x9cMarked Interspecies Variations Concerning the Interactions of Camptothecin with Serum Albumins: A Frequency-Domain Fluorescence Spectroscopic Studyxe2x80x9d, Biochemistry, 33: 12540-12545 (1994). This very attractive feature should greatly facilitate the drug development process and the translation of experimental observations and dosing schedules developed in animal models to the clinic.